Development and validation of a next-generation sequencing assay with open-access analysis software for detecting resistance-associated mutations in CMV.

Cytomegalovirus (CMV) resistance testing by targeted next-generation sequencing (NGS) allows for the simultaneous analysis of multiple genes. We developed and validated an amplicon-based Ion Torrent NGS assay to detect CMV resistance mutations in UL27, UL54, UL56, and UL97 and compared the results to standard Sanger sequencing. NGS primers were designed to generate 83 overlapping amplicons of four CMV genes (~10 kb encompassing 138 mutation sites). An open-access software plugin was developed to perform read alignment, call variants, and interpret drug resistance. Plasmids were tested to determine NGS error rate and minor variant limit of detection. NGS limit of detection was determined using the CMV WHO International Standard and quantified clinical specimens. Reproducibility was also assessed. After establishing quality control metrics, 185 patient specimens previously tested using Sanger were reanalyzed by NGS. The NGS assay had a low error rate (<0.05%) and high accuracy (95%) for detecting CMV-associated resistance mutations present at ≥5% in contrived mixed populations. Mutation sites were reproducibly sequenced with 40× coverage when plasma viral loads were ≥2.6 log IU/mL. NGS detected the same resistance-associated mutations identified by Sanger in 68/69 (98.6%) specimens. In 16 specimens, NGS detected 18 resistance mutations that Sanger failed to detect; 14 were low-frequency variants (<20%), and six would have changed the drug resistance interpretation. The NGS assay showed excellent agreement with Sanger and generated high-quality sequence from low viral load specimens. Additionally, the higher resolution and analytic sensitivity of NGS potentially enables earlier detection of antiviral resistance.

HIV-1 Drug Resistance Assay Using Ion Torrent Next Generation Sequencing and On-Instrument End-to-End Analysis Software.

HIV-1 antiretroviral therapy management requires sequencing the protease, reverse transcriptase, and integrase portions of the HIV-1 pol gene. Most resistance testing is performed with Sanger sequencing, which has limited ability to detect minor variants. Next generation sequencing (NGS) platforms enable variant detection at frequencies as low as 1% allowing for earlier detection of resistance and modification of therapy. Implementation of NGS assays in the clinical laboratory is hindered by complicated assay design, cumbersome wet bench procedures, and the complexity of data analysis and bioinformatics. We developed a complete NGS protocol and companion analysis and reporting pipeline using AmpliSeq multiplex PCR, Ion Torrent S5 XL sequencing, and Stanford's HIVdb resistance algorithm. Implemented as a Torrent Suite software plugin, the pipeline runs automatically after sequencing. An optimum variant frequency threshold of 10% was determined by comparing Sanger sequences of archived samples from ViroSeq testing, resulting in a sensitivity of 98.2% and specificity of 99.0%. The majority (91%) of drug resistance mutations were detected by both Sanger and NGS, with 1.7% only by Sanger and 7.3% only by NGS. Variant calls were highly reproducible and there was no cross-reactivity to VZV, HBV, CMV, EBV, and HCV. The limit of detection was 500 copies/mL. The NGS assay performance was comparable to ViroSeq Sanger sequencing and has several advantages, including a publicly available end-to-end analysis and reporting plugin. The assay provides a straightforward path for implementation of NGS for HIV drug resistance testing in the laboratory setting without additional investment in bioinformatics infrastructure and resources.

Evaluation of the FilmArray® Respiratory Panel for clinical use in a large children's hospital.

BACKGROUND: Respiratory pathogens are a leading cause of hospital admission and traditional detection methods are time consuming and insensitive. Multiplex molecular detection methods have recently been investigated in hope of replacing these traditional techniques with rapid panel-based testing. OBJECTIVES: This study evaluated the FilmArray(®) Respiratory Panel ([FARP], Idaho Technology Inc., Salt Lake City, UT) as a replacement for direct fluorescent antibody (DFA) testing in a pediatric hospital. METHODS: Eleven of the 21 FARP analytes (Adenovirus, Bordetella pertussis, human Metapneumovirus, Influenza A, Influenza A H1N1 2009, Influenza B, Parainfluenza [1, 2, & 3], Respiratory Syncytial Virus, and rhinovirus) were evaluated using nasopharyngeal specimens. Positive samples were pooled in groups of 5. Samples identified by reference methods as positive for respiratory pathogens were used for the majority of positive samples. DFA was the reference method for ten analytes; Luminex™ xTAG Respiratory Virus Panel (RVP) was the reference method for rhinovirus. Discrepant results were resolved by positive culture and fluorescent antibody stain and/or laboratory-developed real-time polymerase chain reaction (PCR) assays (LDT). RESULTS: The agreement for most analytes was in concordance with the established reference methods with the exception of Adenovirus. Additionally, the FARP detected several pathogens not previously detected by DFA, and most were confirmed by LDT. Several DFA-positive analytes were confirmed as true-negatives by the FARP and LDT. CONCLUSION: FARP overall performed better than DFA with the exception of Adenovirus, making the FARP an attractive alternative to laboratories looking to replace DFA with a rapid, user-friendly, multiplex molecular assay.

Limited utility of culture for Mycoplasma pneumoniae and Chlamydophila pneumoniae for diagnosis of respiratory tract infections.

We assessed the utility of culture for Mycoplasma pneumoniae and Chlamydophila pneumoniae to diagnose respiratory tract infections. Compared to PCR and IgM serology, culture was less sensitive and had extremely low yield. Culture is not recommended for these pathogens, and this method should be eliminated from routine practice.

The use of Armored RNA as a multi-purpose internal control for RT-PCR.

Real time reverse transcriptase-PCR (RT-PCR) is now used commonly for the detection of viral pathogens in respiratory samples. However, due to potential inhibition of the RT-PCR or inefficient extraction, this sample type can present significant challenges to accurate patient testing. The goal of this study was to create an internal control to be multiplexed in a real time RT-PCR assay for detecting a viral target in respiratory samples. This report describes an Armored RNA (aRNA) internal control developed originally to be multiplexed in a real time RT-PCR assay for detecting SARS-associated Coronavirus, but can be incorporated into any RT-PCR assay. The internal control primers and probe target a region in the coat protein gene of the E. coli F-specific bacteriophage ms2, which is contained within the aRNA.

Limitations of polymerase chain reaction testing for diagnosing acute Epstein-Barr virus infections.

Clinicians use molecular tests to detect Herpesviridae from blood without fully appreciating limitations of testing. Studies are needed to enhance our understanding of the impact of Herpesviridae latency on molecular testing. We retrospectively performed quantitative Epstein-Barr virus (EBV) on sera from patients between the ages of 1 and 30 who demonstrated serologic evidence of acute EBV (n = 50) or remote EBV (n = 50) infection. Epstein-Barr virus DNA was detected in 70% of acutely infected and 4% of remotely infected patients. Sera from acutely infected patients had higher EBV copy number than convalescent sera. Our results suggest that serology should be performed as the initial diagnostic test for acute EBV. The role for polymerase chain reaction in immunocompromised patients with impaired antibody responses or as a 2nd-line diagnostic test when serologic results are equivocal deserves further study.

Use of modified oligonucleotides to compensate for sequence polymorphisms in the real-time detection of norovirus.

Noroviruses are major causative agents of sporadic and outbreak-associated nonbacterial gastroenteritis worldwide. Real-time RT-PCR has rapidly become the principle means for norovirus detection due to its sensitivity and specificity; however sequence variations in the Norovirus genome can cause problems for real-time chemistries. Using a combination of modified bases and minor groove binder-conjugated Eclipse hybridization probes, a one step real-time RT-PCR assay was designed and optimized to detect norovirus genogroups I and II. Additionally, an RNA internal control was incorporated into the assay to monitor nucleic acid extraction and amplification inhibition. As part of this study, a combination of 36 stool and RNA samples were tested and results were compared to a previously described TaqMan assay. An overall correlation of 97% was obtained. After 7 months of clinical testing, a percent positive rate of 39% was determined, with genogroup II accounting for 98% of the positives. Overall, the Eclipse assay is a sensitive and robust method for detecting and typing norovirus genogroups I and II as well as useful for routine clinical testing. Incorporating modified bases helps overcome design limitations due to single nucleotide polymorphisms.

Expression of estrus before fixed-time AI affects conception rates and factors that impact expression of estrus and the repeatability of expression of estrus in sequential breeding seasons.

Expression of estrus after PG and before fixed-time AI has been reported to change the uterine environment, increase accessory sperm numbers, fertilization rates, and overall embryo survival. Thus, expression of estrus can strongly impact overall pregnancy success. Because of variation in percentage of beef females detected in estrus and number of animals per study, it can be difficult to detect a significant effect of estrus on pregnancy success. Thus, a meta-analysis was conducted using data from 10,116 beef females in 22 studies that utilized variations of the 5 most common fixed-time AI protocols (CO-Synch, 7-day CO-Synch+CIDR, 5-day CIDR, PG 6-day CIDR, and the 14-day CIDR protocols) to examine the effect of detection in standing estrus on subsequent fixed-time AI pregnancy success. A random-effects model was used to combine the studies/herds. The overall model indicated a positive effect of estrus on conception rates with cows detected in estrus before fixed-time AI having a 27% greater (P<0.05; 95% CI=22-32%) conception rate compared with those not detected in estrus. Next we determined factors that influenced expression of estrus. Data were available on 547 cows synchronized with a CIDR based fixed-time AI protocols and observed for estrus before AI during 2-4 breeding seasons. Analysis of these cows indicated that days postpartum (P=0.22) did not impact estrous expression. In contrast, BCS influenced estrous expression (P=0.04) with cows in a BCS of ≤4 (51±5%) having decreased expression of estrus compared to cows with a BCS>4 (≥70±4%). Initiation of estrous cycles before the breeding season also influenced estrous expression (P=0.03), with anestrous cows having greater expression of estrus compared with estrus-cycling cows (78±5% vs. 70±5%, respectively). In conclusion, among all currently recommended fixed-time AI protocols, cows detected in estrus before fixed-time AI had improved conception rates, with BCS and estrus-cycling status having the greatest influence on expression of estrus.

Use of lyophilized standards for the calibration of a newly developed real time PCR assay for human herpes type six (HHV6) variants A and B.

A quantitative real time PCR assay utilizing an Eclipse minor groove binding hybridization probe was developed to detect and type human herpes 6. A 115 base pair product from the U67 gene was selected for amplification and the assay included a noncompetitive internal control. The probe's melting temperature from the amplified sequence differentiated between HHV6 variants (A and B). In this study, 120 samples (60 spiked and 60 negative) comprising CSF, plasma, and serum were tested at high and medium levels, and near the limit of quantitation. The use of stored standard curves for assay calibration was compared to curves run on each assay, and the stability of liquid frozen versus lyophilized frozen stocks for calibrators and controls was assessed. After 9 months of clinical testing, assay performance was examined to determine the percent positive rate and positive sample reproducibility, as well as to evaluate standard curve stability. We obtained 100% correlation to expected results for positive and negative samples. A stored curve proved easier, more cost effective, and more reliable than running a standard curve on each assay. The use of lyophilized standards contributes substantially to the maintenance of reproducible testing over an extended period of time.

A novel capillary electrophoresis-based multiplex PCR assay for detection of respiratory pathogens.

The field of infectious disease testing has recently experienced rapid expansion in the number of multiplexed PCR-based assays available for detecting respiratory pathogens. This study provides a preliminary evaluation of a multiplex assay from Seegene that uses capillary electrophoresis as the detection platform for viral and bacterial respiratory pathogens. We compared this technology to a real-time PCR assay for 3 viral targets. Thirty respiratory samples were collected that had previously tested positive for either Flu A, Flu B, or RSV (ten of each). The Seegene assay detected 9/10 Flu A samples, 9/10 Flu B, and 10/10 RSV, for a total detection rate of 93%. The two samples that were undetected by the Seegene assay both generated late-crossing thresholds on the real-time platform, consistent with low viral loads. The Seeplex assay provides a promising alternative for multiplex respiratory testing.

Description and validation of a novel real-time RT-PCR enterovirus assay.

BACKGROUND: Enteroviruses are a leading cause of aseptic meningitis in adult and pediatric populations. We describe the development of a real-time RT-PCR assay that amplifies a small target in the 5' nontranslated region upstream of the classical Rotbart enterovirus amplicon. The assay includes an RNA internal control and incorporates modified nucleotide chemistry. METHODS: We evaluated the performance characteristics of this design and performed blinded parallel testing on clinical samples, comparing the results with a commercially available RT-PCR assay (Pan-Enterovirus OligoDetect kit) that uses an enzyme immunoassay-like plate end detection. RESULTS: We tested 778 samples and found 14 discrepant samples between the 2 assays. Of these, the real-time assay detected 6 samples that were negative by the OligoDetect kit, 5 of which were confirmed as positive by sequence analysis using an alternative primer set. Eight discrepant samples were positive by the OligoDetect kit and real-time negative, with 6 confirmed by sequencing. Overall, detection rates of 97% and 96% were obtained for the OligoDetect kit and real-time assays, respectively. Sequence analysis revealed the presence of a number of single nucleotide polymorphisms in the targeted region. The comparative sensitivities of the 2 assays were equivalent, with the limit of detection for the real-time assay determined to be approximately 430 copies per milliliter in cerebrospinal fluid. CONCLUSIONS: This novel real-time enterovirus assay is a sensitive and suitable assay for routine clinical testing. The presence of single nucleotide polymorphisms can affect real-time PCR assays.

Evaluation of quantification methods for real-time PCR minor groove binding hybridization probe assays.

Real-time PCR data analysis for quantification has been the subject of many studies aimed at the identification of new and improved quantification methods. Several analysis methods have been proposed as superior alternatives to the common variations of the threshold crossing method. Notably, sigmoidal and exponential curve fit methods have been proposed. However, these studies have primarily analyzed real-time PCR with intercalating dyes such as SYBR Green. Clinical real-time PCR assays, in contrast, often employ fluorescent probes whose real-time amplification fluorescence curves differ from those of intercalating dyes. In the current study, we compared four analysis methods related to recent literature: two versions of the threshold crossing method, a second derivative maximum method, and a sigmoidal curve fit method. These methods were applied to a clinically relevant real-time human herpes virus type 6 (HHV6) PCR assay that used a minor groove binding (MGB) Eclipse hybridization probe as well as an Epstein-Barr virus (EBV) PCR assay that used an MGB Pleiades hybridization probe. We found that the crossing threshold method yielded more precise results when analyzing the HHV6 assay, which was characterized by lower signal/noise and less developed amplification curve plateaus. In contrast, the EBV assay, characterized by greater signal/noise and amplification curves with plateau regions similar to those observed with intercalating dyes, gave results with statistically similar precision by all four analysis methods.

Effect of sequence polymorphisms on performance of two real-time PCR assays for detection of herpes simplex virus.

Herpes simplex virus (HSV) is the most common cause of acquired, sporadic encephalitis in the United States. PCR identification of HSV in spinal fluid has become the diagnostic gold standard due to its sensitivity and potential for speed, replacing other methods such as culture. We developed a real-time PCR assay to detect HSV, using a new type of hybridization probe, the Eclipse probe. In this study, we ran 323 samples (171 positives and 152 negatives) with the Eclipse real-time PCR assay and compared these results with another PCR assay using gel detection. The real-time assay agreed with our reference method for 319 out of the 323 samples tested (99%). Using two different real-time PCR platforms, we discovered that SNPs within the amplicon's probe binding region that are used to distinguish HSV-1 from HSV-2 can decrease assay sensitivity. This problem is potentially a general one for assays using fluorescent probes to detect target amplification in a real-time format. While real-time PCR can be a powerful tool in the field of infectious disease, careful sequence evaluation and clinical validation are essential in creating an effective assay.